Leukemias are malignant neoplasms of hematopoietic tissues. These neoplasms are categorized into two predominant forms: chronic and acute. While acute leukemias are characterized by undifferentiated cell populations, chronic leukemias usually present a more mature morphology. In general the pathological impairment of normal hematopoiesis is the hallmark of all leukemias
A tight control of kinase and phosphatase activity is fundamental for normal cell growth, survival, and differentiation. Leukemia is often associated with expression of oncoproteins with aberrant kinase activity. By contrast, PP2A, a phosphatase regulating many cellular functions, is genetically or functionally inactivated in many types of cancer.
Suppression of PP2A activity appears to be a common event in malignant transformation. Moreover, cellular transformation by the SV40 viral oncoprotein small t Ag requires inactivation of PP2A (Yang et al., Control of protein phosphatase 2A by simian virus 40 small-t antigen, Mol. Cell. Biol. 11 (1991), pp. 1988-1995), and PP2Ac overexpression reduces Ha-RAS-induced cell transformation (Z. Baharians and A. H. Schonthal, Reduction of Ha-ras-induced cellular transformation by elevated expression of protein phosphatase type 2A, Mol. Carcinog. 24 (1999), pp. 246-254). The role of PP2A depends on its ability to interact with and dephosphorylate several factors implicated in the regulation of cell cycle progression, proliferation, survival, and differentiation (E. Sontag, Protein phosphatase 2A: the Trojan Horse of cellular signaling, Cell. Signal. 13 (2001), pp. 7-16). The catalytic subunit of PP2A has been linked to retinoic acid-induced cellular differentiation of HL-60 cells, an acute promyelocytic leukemia (APL) cell line (Nishikawa et al., Cancer Res., 1994, 54, 4879-4884; Tawara et al., FEBS Lett., 1993, 321, 224-228).
Forskolin is the chemical active ingredient extracted from the roots of Coleus Forskohlii. This natural product has been traditionally used in Hindu traditional medicine to treat asthma, heart disease, glaucoma and more. It is currently used in Japan to treat patients with cardiomyopathy and brochospasm. In addition, there are several lines of evidence showing the in vivo and in vitro anti-tumor activity of this compound against several solid tumor and leukemic cell lines. Forskolin, a diterpene, is a potent activator of adenylate cyclase that has been used extensively to increase intracellular cAMP levels and to elicit cAMP-dependent physiological responses. (e.g. WO 2004/062671). Others have reported the synergistic activity of forskolin, as a known phosphatase kinase A (PKA) agonist, with retinoid X or retinoid acid receptor agonists useful in the treatment of APL or breast cancer. (e.g. U.S. Pat. No. 6,624,154).
Chronic Myelogenous Leukemia (CML)
CML arises when two chromosomes, 9 and 22, mistakenly exchange genetic material during cell division. This translocation t(9;22)(q34;q11), designated as the Philadelphia chromosome (Ph1), creates a new, fused gene (called BCR-ABL), that produces an oncogenic enzyme called Bcr-Abl. Bcr-Abl permanently turns on cell growth signals that are normally held in check by phosphatases, and the result is the uncontrolled production of white blood cells.
The clinical course of CML involves the progression from a stable syndrome “chronic phase” (CML-CP) to an acute and fatal stage “blast crisis” (CML-BC) marked by the clonal expansion of an immature population of differentiation-arrested myeloid blasts. BCR/ABL induces and sustains the leukemic phenotype through its deregulated tyrosine kinase activity, which is essential for the recruitment and activation of multiple pathways that transduce oncogenic signals leading to growth factor-independent proliferation, increased survival and altered differentiation of myeloid precursors. Dependence on BCR/ABL expression is not only a characteristic of CML-CP; in fact, levels of BCR/ABL often increase during disease progression and sustained BCR/ABL expression in myeloid progenitor cell lines induces phenotypic changes (i.e. suppression of granulocytic differentiation) characteristic of blast crisis CML.
The goal in treating CML is to eliminate cells containing the Philadelphia chromosome, achieving a complete remission. Stem cell transplantation, when possible, has been the therapy most likely to enable patients with CML to achieve long-term complete remissions. In the past, treatments regimens included chemotherapy (such as with interferon-alpha, hydroxyurea, busulfan). Nowadays the main treatment option for CML patients is imatinib mesylate (Gleevec™, formerly STI571, Novartis). Other potential treatments include dasatinib (Bristol-Myers Squibb, formerly known as BMS-354825) and AMN107 (Novartis). Specific inhibition of BCR/ABL kinase activity with imatinib mesylate is effective not only in the therapy of chronic phase CML but also, albeit temporarily, of accelerate and blastic phase CML, in which imatinib resistance and relapses are contingent to reactivation of BCR/ABL activity. Since this therapy is only a few years old, it is unknown at this time if the complete remissions achieved with this therapy will be as long lasting as the cases after successful stem cell transplant and if progression of CML into blast crisis is prevented.
The main problem with imatinib treatment is the development of resistance and relapse of the disease, which at this moment is considered a factor that favors progression into the fatal blast crisis stage. Main mechanisms of resistance are point mutations, amplification and increased expression of the BCR/ABL oncogene. One of the more problematic mutations is the BCR/ABL T315I mutation, and it is responsible for about 15 percent of cases in which CML patients develop resistance to imatinib. The T315I mutation also confers resistance to dasatinib and AMN107. Dasatinib and AMN107, reportedly can overcome some more than fifty mutations that cause resistance to imatinib, with the exception of T315I. So, it is of particular importance to find a drug that can treat patients with CML harboring the T315I BCR/ABL mutant.
Philadelphia Positive Acute Lymphoblastic Leukemia (Ph1-ALL)
It has been recognized for many years that some patients presenting with acute leukemia may have a cytogenetic abnormality that is morphologically indistinguishable from the Philadelphia chromosome (Ph1). (See Peterson L C, Bloomfield C D, Bruning R D: Blast crisis as an initial or terminal manifestation of chronic myeloid leukemia: a study of 28 patients. Am J Med 60(2): 209-220, 1976.) The Ph1 occurs in about 30% of adults and a small percentage of children with ALL. (See Seeker-Walker L M, et al.: Variable Philadelphia breakpoints and potential lineage restriction of bcr rearrangement in acute lymphoblastic leukemia. Blood 72 (2): 784-91, 1988.) In the majority of children and in more than one half of adults with Ph1-positive ALL, the molecular abnormality (known as p190 BCR/ABL) is different from that in Ph1-positive CML (known as p210 BCR/ABL).
The Ph1 chromosome may be detected using cytogenetic methods to find the chromosome and the translocation. However, many patients who have molecular evidence of the BCR/ABL fusion gene, which characterizes the Ph1, have no evidence of the abnormal chromosome by cytogenetics. Because many patients have a different fusion protein from the one found in CML (p190 versus p210), the BCR/ABL fusion gene may be detectable only by pulsed-field gel electrophoresis or reverse-transcriptase polymerase chain reaction (RT-PCR). These tests should be performed whenever possible in patients with ALL, especially those with B-cell lineage disease. BCR/ABL rearranged leukemias that do not demonstrate the classical Ph1 carry a poor prognosis that is similar to those that are Ph1-positive. (See Chromosomal abnormalities and their clinical significance in acute lymphoblastic leukemia. Third International Workshop on Chromosomes in Leukemia. Cancer Res 43 (2): 868-73, 1983.)
Ph1-ALL represents 40% of ALL cases in patients over the age of 40 years. Adults with Ph1-ALL, have a poor prognosis and survival at 3 years does not exceed 20% in most studies.
There is a great need for the development of agents for the treatment of BCR/ABL-mediated leukemia patients, particularly those who have developed resistance to therapy, those in the advanced stages of CML or for those with Ph1-ALL.